Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

Abstract

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [ 3H]dThd was lower than that of [5- 3H]dCyd, but almost all of [ 3H]dThd was incorporated into DNA. However, the main part of [5- 3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [ 3H]dThd incorporation, while [5- 3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [ 3H]dThd was in dTMP form, while [5- 3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.